Multiplex Detection
Large-scale multiplex analysis of highly polymorphic loci is needed for practical identification of individuals, e.g., for paternity testing and in forensic science (Reynolds et al., Anal. Chem., 63:2-15 (1991)), for organ-transplant donor-recipient matching (Buyse et al., Tissue Antigens, 41:1-14 (1993) and Gyllensten et al., PCR Meth. Appl, 1:91-98 (1991)), for genetic disease diagnosis, prognosis, and pre-natal counseling (Chamberlain et al., Nucleic Acids Res., 16:11141-11156 (1988) and L. C. Tsui, Human Mutat., 1:197-203 (1992)), and the study of oncogenic mutations (Hollstein et al., Science, 253:49-53 (1991)). In addition, the cost-effectiveness of infectious disease diagnosis by nucleic acid analysis varies directly with the multiplex scale in panel testing. Many of these applications depend on the discrimination of single-base differences at a multiplicity of sometimes closely spaced loci.
A variety of DNA hybridization techniques are available for detecting the presence of one or more selected polynucleotide sequences in a sample containing a large number of sequence regions. In a simple method, which relies on fragment capture and labeling, a fragment containing a selected sequence is captured by hybridization to an immobilized probe. The captured fragment can be labeled by hybridization to a second probe which contains a detectable reporter moiety.
Another widely used method is Southern blotting. In this method, a mixture of DNA fragments in a sample is fractionated by gel electrophoresis, then fixed on a nitrocellulose filter. By reacting the filter with one or more labeled probes under hybridization conditions, the presence of bands containing the probe sequences can be identified. The method is especially useful for identifying fragments in a restriction-enzyme DNA digest which contains a given probe sequence and for analyzing restriction-fragment length polymorphisms ("RFLPs").
Another approach to detecting the presence of a given sequence or sequences in a polynucleotide sample involves selective amplification of the sequence(s) by polymerase chain reaction. U.S. Pat. No. 4,683,202 to Mullis, et al. and R. K. Saiki, et al., Science 230:1350 (1985). In this method, primers complementary to opposite end portions of the selected sequence(s) are used to promote, in conjunction with thermal cycling, successive rounds of primer-initiated replication. The amplified sequence(s) may be readily identified by a variety of techniques. This approach is particularly useful for detecting the presence of low-copy sequences in a polynucleotide-containing sample, e.g., for detecting pathogen sequences in a body-fluid sample.
More recently, methods of identifying known target sequences by probe ligation methods have been reported. U.S. Pat. No. 4,883,750 to N. M. Whiteley, et al., D. Y. Wu, et al., Genomics 4:560 (1989), U. Landegren, et al., Science 241:1077 (1988), and E. Winn-Deen, et al., Clin. Chem. 37:1522 (1991). In one approach, known as oligonucleotide ligation assay ("OLA"), two probes or probe elements which span a target region of interest are hybridized to the target region. Where the probe elements basepair with adjacent target bases, the confronting ends of the probe elements can be joined by ligation, e.g., by treatment with ligase. The ligated probe element is then assayed, indicating the presence of the target sequence.
In a modification of this approach, the ligated probe elements act as a template for a pair of complementary probe elements. With continued cycles of denaturation, hybridization, and ligation in the presence of pairs of probe elements, the target sequence is amplified linearly, allowing very small amounts of target sequence to be detected and/or amplified. This approach is referred to as ligase detection reaction. When two complementary pairs of probe elements are utilized, the process is referred to as the ligase chain reaction which achieves exponential amplification of target sequences. F. Barany, "Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase," Proc. Nat'l Acad. Sci. USA, 88:189-93 (1991) and F. Barany, "The Ligase Chain Reaction (LCR) in a PCR World," PCR Methods and Applications, 1:5-16 (1991).
Another scheme for multiplex detection of nucleic acid sequence differences is disclosed in U.S. Pat. No. 5,470,705 to Grossman et. al. where sequence-specific probes, having a detectable label and a distinctive ratio of charge/translational frictional drag, can be hybridized to a target and ligated together. This technique was used in Grossman, et. al., "High-density Multiplex Detection of Nucleic Acid Sequences: Oligonucleotide Ligation Assay and Sequence-coded Separation," Nucl. Acids Res. 22(21): 4527-34 (1994) for the large scale multiplex analysis of the cystic fibrosis transmembrane regulator gene.
Jou, et. al., "Deletion Detection in Dystrophia Gene by Multiplex Gap Ligase Chain Reaction and Immunochromatographic Strip Technology," Human Mutation 5:86-93 (1995) relates to the use of a so called "gap ligase chain reaction" process to amplify simultaneously selected regions of multiple exons with the amplified products being read on an immunochromatographic strip having antibodies specific to the different haptens on the probes for each exon.
There is a growing need (e.g., in the field of genetic screening) for methods useful in detecting the presence or absence of each of a large number of sequences in a target polynucleotide. For example, as many as 400 different mutations have been associated with cystic fibrosis. In screening for genetic predisposition to this disease, it is optimal to test all of the possible different gene sequence mutations in the subject's genomic DNA, in order to make a positive identification of "cystic fibrosis". It would be ideal to test for the presence or absence of all of the possible mutation sites in a single assay. However, the prior-art methods described above are not readily adaptable for use in detecting multiple selected sequences in a convenient, automated single-assay format.
Solid-phase hybridization assays require multiple liquid-handling steps, and some incubation and wash temperatures must be carefully controlled to keep the stringency needed for single-nucleotide mismatch discrimination. Multiplexing of this approach has proven difficult as optimal hybridization conditions vary greatly among probe sequences.
Developing a multiplex PCR process that yields equivalent amounts of each PCR product can be difficult and laborious. This is due to variations in the annealing rates of the primers in the reaction as well as varying polymerase extension rates for each sequence at a given Mg.sup.2+ concentration. Typically, primer, Mg.sup.2+, and salt concentrations, along with annealing temperatures are adjusted in an effort to balance primer annealing rates and polymerase extension rates in the reaction. Unfortunately, as each new primer set is added to the reaction, the number of potential amplicons and primer dimers which could form increases exponentially. Thus, with each added primer set, it becomes increasingly more difficult and time consuming to work out conditions that yield relatively equal amounts of each of the correct products.
Allele-specific PCR products generally have the same size, and an assay result is scored by the presence or absence of the product band(s) in the gel lane associated with each reaction tube. Gibbs et al., Nucleic Acids Res., 17:2437-48 (1989). This approach requires splitting the test sample among multiple reaction tubes with different primer combinations, multiplying assay cost. In PCR, discrimination of alleles can be achieved by attaching different fluorescent dyes to competing allelic primers in a single reaction tube (F. F. Chehab, et al., Proc. Natl. Acad. Sci. USA, 86:9178-9182 (1989)), but this route to multiplex analysis is limited in scale by the relatively few dyes which can be spectrally resolved in an economical manner with existing instrumentation and dye chemistry. The incorporation of bases modified with bulky side chains can be used to differentiate allelic PCR products by their electrophoretic mobility, but this method is limited by the successful incorporation of these modified bases by polymerase, and by the ability of electrophoresis to resolve relatively large PCR products which differ in size by only one of these groups. Livak et al., Nucleic Acids Res., 20:4831-4837 (1989). Each PCR product is used to look for only a single mutation, making multiplexing difficult.
Ligation of allele-specific probes generally has used solid-phase capture (U. Landegren et al., Science, 241:1077-1080 (1988); Nickerson et al., Proc. Natl. Acad. Sci. USA, 87:8923-8927 (1990)) or size-dependent separation (D. Y. Wu, et al., Genomics, 4:560-569 (1989) and F. Barany, Proc. Natl. Acad. Sci., 88:189-193 (1991)) to resolve the allelic signals, the latter method being limited in multiplex scale by the narrow size range of ligation probes. Further, in a multiplex format, the ligase detection reaction alone cannot make enough product to detect and quantify small amounts of target sequences. The gap ligase chain reaction process requires an additional step--polymerase extension. The use of probes with distinctive ratios of charge/translational frictional drag for a more complex multiplex process will either require longer electrophoresis times or the use of an alternate form of detection.
The need thus remains for a rapid single assay format to detect the presence or absence of multiple selected sequences in a polynucleotide sample when those sequences are in low abundance. Such detection is required when cancer-associated mutations are present in an excess of normal cells.
DNA Ligase
DNA ligases catalyze the formation of phosphodiester bonds at single-stranded breaks (nicks) in double-stranded DNA, and are required in DNA replication, repair, and recombination. The general mechanism of ligation reactions involves three reversible steps, as shown below for NAD.sup.+ -dependent ligases. In this example, the nicked DNA substrate is formed by annealing two short oligonucleotides (oligo A and B) to a longer complementary oligonucleotide. First, a covalently adenylated enzyme intermediate is formed by transfer of the adenylate group of NAD.sup.+ to the e-NH2 group of lysine in the enzyme. Second, the adenylate moiety is transferred from the enzyme to the 5'-terminal phosphate on oligo B. Finally, a phosphodiester bond is formed by a nucleophilic attack of the 3'-hydroxyl terminus of oligo A on the activated 5'-phosphoryl group of oligo B (Gumport, R. I., et al., Proc. Natl. Acad. Sci. USA, 68:2559-63 (1971); Modrich, P., et al., J. Biol. Chem., 248:7495-7501 (1973); Modrich, P., et al., J. Biol. Chem., 248:7502-11 (1973); Weiss, B., et al., J. Biol. Chem., 242:4270-72 (1967); Weiss, B., et al., J. Biol. Chem., 243:4556-63 (1968); Becker, A., et al., Proc. Natl. Acad. Sci. USA, 58:1996-2003 (1967); Yudelevich, A., et al., Proc. Natl. Acad. Sci. USA, 61:1129-36 (1968); Zimmerman, S. B., et al., Proc. Natl. Acad. Sci. USA, 57:1841-48 (1967); Zimmerman, S. B., et al., J. Biol. Chem., 244:4689-95 (1969); and Lehman, I. R., Science, 186:790-97 (1974)).
(i) E-(lys)-NH.sub.2 +AMP.about.PRN.sup.+ {character pullout}E-(lys)-NH.sub.2.sup.+.about.AMP+NMN PA1 (ii) E-(lys)-NH.sub.2.sup.+.about.AMP+5'P-Oligo B{character pullout}+AMP.about.P-Oligo B+E-(lys)-NH.sub.2 PA1 (iii) Oligo A-3'OH+AMP.about.P-Oligo B{character pullout}Oligo A-P-Oligo B+AMP
Within the last decade, genes encoding ATP-dependent DNA ligases have been cloned and sequenced from bacteriophages T3, T4, and T7 (Dunn, J. J., et al., J. Mol. Biol., 148:303-30 (1981); Armstrong, J., et al., Nucleic Acids Res., 11:7145-56 (1983); and Schmitt, M. P., et al., J. Mol. Biol., 193:479-95 (1987)), African swine fever virus (Hammond, J. M., et al., Nucleic Acids Res., 20:2667-71 (1992)), Vaccinia virus (Smith, G. L., et al., Nucleic Acids Res., 17:9051-62 (1989)), Shope fibroma virus (Parks, R. J., et al., Virology, 202:642-50 (1994)), an extremely thermophilic archaeon Desulfurolobus ambivalens (Kletzin, A., Nucleic Acids Res., 20:5389-96 (1992)), S. cerevisiae (CDC9 gene)(Barker, D. G., et al., Mol. Gen. Genet., 200:458-62 (1985)); S. pombe (cdcl7+) (Barker, D. G., et al., Eur. J. Biochem., 162:659-67 (1988)), Xiphophorus (Walter, R. B., et al., Mol. Biol. Evol., 10:1227-38 (1993)); mouse fibroblast (Savini, E., et al., Gene, 144:253-57 (1994)); and Homo sapiens (human DNA ligase I, III, and IV) (Barnes, D. E., et al., Proc. Natl. Acad. Sci. USA, 87:6679-83 (1990); Chen, J., et al., Molec. and Cell. Biology, 15:5412-22 (1995); and Wei, Y. F., et al., Molec. & Cell. Biology, 15:3206-16 (1995)). In addition, five NAD.sup.+ -dependent bacterial DNA ligases have also been cloned: E. coli (Ishino, Y., et al., Mol. Gen. Genet., 204:1-7 (1986)), Zymomonas mobilis (Shark, K. B., et al., FEMS Microbiol. Lett., 96:19-26 (1992)), Thermus thermophilus (Barany, F., et al., Gene, 109:1-11 (1991) and Lauer, G., et al., J. Bacteriol., 173:5047-53 (1991)), Rhodothermus marinus (Thorbjarnardottir, S. H., et al., Gene, 161:1-6 (1995)), and Thermus scotoductus (Jonsson, Z. O., et al., Gene, 151:177-80 (1994)). ATP-dependent DNA ligases, as well as mammalian DNA ligases I and II contain a conserved active site motif, K(Y/A) DGXR, which includes the lysine residue that becomes adenylated (Tomkinson, A. E., et al, Proc. Natl. Acad. Sci. USA, 88:400-04 (1991) and Wang, Y. C., et al., J. Biol. Chem., 269:31923-28 (1994)). NAD.sup.+ -dependent bacterial DNA ligases contain a similar active site motif, KXDG, whose importance is confirmed in this work.
In vitro experiments using plasmid or synthetic oligonucleotide substrates reveal that T4 DNA ligase exhibits a relaxed specificity; sealing nicks with 3'- or 5'-AP sites (apurinic or apyrimidinic) (Goffin, C., et al., Nucleic Acids Res., 15(21): 8755-71 (1987)), one-nucleotide gaps (Goffin, C., et al., Nucleic Acids Res., 15(21): 8755-71 (1987)), 3'- and 5'-A-A or T-T mismatches (Wu, D. Y., et al., Gene, 76:245-54 (1989)), 5'-G-T mismatches (Harada, K., et al., Nucleic Acids Res., 21(10): 2287-91 (1993)), 3'-C-A, C-T, T-G, T-T, T-C, A-C, G-G, or G-T mismatches (Landegren, U., et al., Science, 241:1077-80 (1988)). The apparent fidelity of T4 DNA ligase may be improved in the presence of spermidine, high salt, and very low ligase concentration, where only T-G or G-T mismatch ligations were detected (Wu, D. Y., et al., Gene, 76:245-54 (1989) and Landegren, U., et al., Science, 241:1077-80 (1988)). DNA ligase from Saccharomyces cerevisiae discriminates 3'-hydroxyl and 5'-phosphate termini separated by a one-nucleotide gap and 3'-A-G or T-G mismatches, however 5'-A-C, T-C, C-A, or G-A mismatches had very little effect on ligation efficiency (Tomkinson, A. E., et al., Biochemistry, 31:11762-71 (1992)). Mammalian DNA ligases I and III show different efficiencies in ligating 3.degree. C.-T, G-T, and T-G mismatches (Husain, I., et al., J. Biol. Chem., 270:9638-90 (1995)). The Vaccinia DNA virus efficiently discriminates against one-nucleotide, two-nucleotide gaps and 3'-G-A, A-A, G-G, or A-G (purine-purine) mismatches, but easily seals 5'-C-T, G-T, T-T, A-C, T-C, C-C, G-G, T-G, or A-G mismatches as well as 3'-C-A, C-T, G-T, T-T, or T-G mismatches (Shuman, S., Biochem., 34:16138-47 (1995)).
The thermostable Thermus thermophilus DNA ligase (Tth DNA ligase) has been cloned and used in the ligase chain reaction (LCR) and ligase detection reaction (LDR) for detecting infectious agents and genetic diseases (Barany, F., Proc. Natl. Acad. Sci. USA, 88:189-93 (1991); Day, D., et al., Genomics, 29:152-62 (1995); Eggerding, F. A., PCR Methods and Applications, 4:337-45 (1995); Eggerding, F. A., et al., Human Mutation, 5:153-65 (1995); Feero, W., et al., Neurology, 43:668-73 (1993); Frenkel, L. M., et al., J. Clin. Micro., 33(2): 342-47 (1995); Grossman, P. D., et al., Nucleic Acids Res., 22:4527-34 (1994); lovannisci, D. M., et al., Mol. Cell. Probes, 7:35-43 (1993); Prchal, J. T., et al., Blood, 81:269-71 (1993); Ruiz-Opaz, N., et al., Hypertension, 24:260-70 (1994); Wiedmann, M., et al., Appl. Environ. Microbiol., 58:3443-47 (1992); Wiedmann, M., et al., Appl Environ Microbiol, 59(8): 2743-5 (1993); Winn-Deen, E., et al., Amer. J. Human Genetics, 53:1512 (1993); Winn-Deen, E. S., et al., Clin. Chem., 40:1092 (1994); and Zebala, J., et al., "Detection of Leber's Hereditary Optic Neuropathy by nonradioactive-LCR. PCR Strategies," (Innis, M. A., Gelfand, D. H., and Sninsky, J. J., Eds.), Academic Press, San Diego (1996)). The success of these and future disease detection assays, such as identifying tumor associated mutations in an excess of normal DNA, depend on the exquisite fidelity of Tth DNA ligase.
Cancer Detection
As the second leading cause of death in this country, almost 600,000 people will die from cancer per year making cancer one of the most alarming of all medical diagnosis. Lifetime risks for developing invasive cancers in men and women are 50 percent and 33 percent, respectively. Expectations are that more than 1.2 million new cases of cancer will be diagnosed in the United States in 1995. Healthcare expenses for cancer in 1994 were approximately $104 billion. However, the full impact of cancer on families and society is not measured only by the amount of money spent on its diagnosis and treatment. A significant number of people are stricken with cancer in their most productive years. Cancers accounted for 18 percent of premature deaths in 1985 and in 1991 more than 9,200 women in the U.S. died from breast cancer before the age of 55. For colorectal and breast cancers, estimates are that nearly 140,000 and 183,000 new diagnoses, respectively, are predicted for 1995.
Currently, diagnosis of cancer is based on histological evaluation of tumor tissue by a pathologist. After a cancer is diagnosed, treatment is determined primarily by the extent or stage of the tumor. Tumor stage is defined by clinical, radiological, and laboratory methods. Standardized classification systems for the staging of tumors have been developed to clearly convey clinical information about cancer patients. Staging provides important prognostic information and forms the basis of clinical studies which allow the testing of new treatment strategies. A staging system was developed (TNM staging system), which classifies tumors according to the size of the primary tumor, the number of regional lymph nodes in which cancer is found, and the presence or absence of metastases to other parts of the body. Smaller cancers with no affected lymph nodes and no distant metastases are considered early stage cancers, which are often amenable to cure through surgical resection. A common measure of prognosis is the 5-year survival rate, the proportion of patients alive five years after the diagnosis of a cancer at a given stage. While 5-year survival rates for many cancers have improved over the last few decades, the fact that some early stage cancers recur within five years or later has led researchers to explore other additional prognostic markers including histological grade, cytometry results, hormone receptor status, and many other tumor markers. Most recently, investigators have explored the use of molecular alterations in cancers as markers of prognosis.
Genetic alterations found in cancers, such as point mutations and small deletions mentioned above, can act as markers of malignant cells.
Detection of Minority Nucleic Acid Sequences
A number of procedures have been disclosed to detect cancer using PCR. Sidransky, et. al., "Identification of ras Oncogene Mutations in the Stool of Patients with Curable Colorectal Tumors," Science 256: 102-05 (1992) detects colon cancer by identification of K-ras mutations. This involves a PCR amplification of total DNA, cloning into a phage vector, plating out the phage, repeated probing with individual oligonucleotides specific to several different K-ras mutations, and counting the percentage of positive plaques on a given plate. This is a technically difficult procedure which takes three days to complete, whereby the ratio of mutant to wild-type DNA in the stool sample is determined. Brennan, et. al., "Molecular Assessment of Histopathological Staging in Squamous-Cell Carcinoma of the Head and Neck," N. Engl. J. Med. 332(7): 429-35 (1995) finds p53 mutations by sequencing. This specific mutation is then probed for in margin tissue using PCR amplification of total DNA, cloning into a phage vector, plating out the phage, probing with an individual oligonucleotide specific to the mutation found by sequencing, and counting the percentage of positive plaques on a given plate. Berthelemy, et. al., "Brief Communications--Identification of K-ras Mutations in Pancreatic Juice in the Early Diagnosis of Pancreatic Cancer," Ann. Int. Med. 123(3): 188-91 (1995) uses a PCR/restriction enzyme process to detect K-ras mutations in pancreatic secretions. This technique is deficient, however, in that mutations are not quantified. Similarly, Tada, et. al., "Detection of ras Gene Mutations in Pancreatic Juice and Peripheral Blood of Patients with Pancreatic Adenocarcinoma," Cancer Res. 53: 2472-74 (1993) and Tada, et. al., "Clinical Application of ras Gene Mutation for Diagnosis of Pancreatic Adenocarcinoma," Gastroent. 100: 233-38 (1991) subject such samples to allele-specific PCR to detect pancreatic cancer. This has the disadvantages of providing false positives due to polymerase extension off normal template, requiring electrophoretical separation of products to distinguish from primer dimers, being unable to multiplex closely-clustered sites due to interference of overlapping primers, being unable to detect single base or small insertions and deletions in small repeat sequences, and not being ideally suitable for quantification of mutant DNA in a high background of normal DNA. Hayashi, et. al., "Genetic Detection Identifies Occult Lymph Node Metastases Undetectable by the Histopathological Method," Cancer Res. 54: 3853-56 (1994) uses an allele-specific PCR technique to find K-ras or p53 mutations to identify occult lymph node metastases in colon cancers. A sensitivity of one tumor cell in one thousand of normal cells is claimed; however, obtaining quantitative values requires laborious cloning, plating, and probing procedures. In Mitsudomi, et. al., "Mutations of ras Genes Distinguish a Subset of Non-small-cell Lung Cancer Cell Lines from Small-cell Lung Cancer Cell Lines," Oncogene 6: 1353-62 (1991), human lung cancer cell lines are screened for point mutations of the K-, H-, and N-ras genes using restriction fragment length polymorphisms created through mismatched primers during PCR amplification of genomic DNA. The disadvantages of such primer-mediated RFLP include the requirement of electrophoretical separation to distinguish mutant from normal DNA, limited applicability to sites that may be converted into a restriction site, the requirement for additional analysis to determine the nature of the mutation, and the difficulty in quantifying mutant DNA in a high background of normal DNA. Further, these procedures tend to be laborious and inaccurate.
Coupled PCR/ligation processes have been used for detection of minority nucleotide sequences in the presence of majority nucleotide sequences. A PCR/LDR process is used in Frenkel, "Specific, Sensitive, and Rapid Assay for Human Immunodeficiency Virus Type 1 pol Mutations Associated with Resistance to Zidovudine and Didanosine," J. Clin. Microbiol. 33(2): 342-47 (1995) to detect HIV mutants. This assay, however, cannot be used for multiplex detection. See also Abravaya, et. al., "Detection of Point Mutations With a Modified Ligase Chain (Gap-LCR)," Nucl. Acids Res. 23(4): 675-82 (1995) and Balles, et. al., "Facilitated Isolation of Rare Recombinants by Ligase Chain Reaction: Selection for Intragenic Crossover Events in the Drosophila optomotor-blind Gene," Molec. Gen. Genet. 245: 734-40 (1994).
Colorectal lesions have been detected by a process involving PCR amplification followed by an oligonucleotide ligation assay. See Jen, et. al., "Molecular Determinants of Dysplasia in Colorectal Lesions," Cancer Res. 54: 5523-26 (1994) and Redston, et. al., "Common Occurrence of APC and K-ras Gene Mutations in the Spectrum of Colitis-Associated Neoplasias," Gastroenter. 108: 383-92 (1995). This process was developed as an advance over Powell, et. al., "Molecular Diagnosis of Familial Adenomatous Polyposis," N. Engl. J. Med. 329(27): 1982-87 (1993). These techniques tend to be limited and difficult to carry out.
Other procedures have been developed to detect minority nucleotide sequences. Lu, et. al., "Quanititative Aspects of the Mutant Analysis by PCR and Restriction Enzyme Cleavage (MAPREC)" PCR Methods and Appl. 3: 176-80 (1993) detects virus revertants by PCR and restriction enzyme cleavage. The disadvantages of MAPREC include the requirement for electrophoretical separation to distinguish mutant from normal DNA, limited applicability to sites that may be converted into a restriction site, the requirement for additional analysis to determine the nature of the mutation, and difficulty in quantifying mutant DNA in a high background of normal DNA. In Kuppuswamy, et. al., "Single Nucleotide Primer Extension to Detect Genetic Diseases: Experimental Application to Hemophilia G (Factor IX) and Cystic Fibrosis Genes," Proc. Natl. Acad. Sci. USA 88: 1143-47 (1991), a PCR process is carried out using 2 reaction mixtures for each fragment to be amplified with one mixture containing a primer and a labeled nucleotide corresponding to the normal coding sequence, while the other mixture contains a primer and a labeled nucleotide corresponding to the mutant sequence. The disadvantages of such mini sequencing (i.e. SNuPe) are that the mutations must be known, it is not possible to multiplex closely clustered sites due to interference of overlapping primers, it is not possible to detect single base or small insertions and deletions in small repeat sequences, and four separate reactions are required. A mutagenically separated PCR process is disclosed in Rust, et. al., "Mutagenically Separated PCR (MS-PCR): a Highly Specific One Step Procedure for easy Mutation Detection" Nucl. Acids Res. 21(16): 3623-29 (1993) to distinguish normal and mutant alleles, using different length allele-specific primers. The disadvantages of MS-PCR include possibly providing false positives due to polymerase extension off normal template, requiring electrophoretical separation of products to distinguish from primer dimers, the inability to multiplex closely-clustered sites due to interference of overlapping primers, the inability to detect single base or small insertions and deletions in small repeat sequences, and not being ideally suited for quantification of mutant DNA in high background of normal DNA. In Suzuki, et. al., "Detection of ras Gene Mutations in Human Lung Cancers by Single-Strand Conformation Polymorphism Analysis of Polymerase Chain Reaction Products," Oncogene 5: 1037-43 (1990), mutations are detected in a process having a PCR phase followed by phase involving single strand conformation polymorphism ("SSCP") of the amplified DNA fragments. The disadvantages of SSCP include the requirement for electrophoretical separation to distinghish mutant conformer from normal conformer, the failure to detect 30% of possible mutations, the requirement for additional analysis to determine the nature of the mutation, and the inability to distinguish mutant from silent polymorphisms.
Despite the existence of techniques for detecting minority nucleotide sequences in the presence of majority sequences, the need remains for improved procedures of doing so. It is particularly desirable to develop such techniques where minority nucleotide sequences can be quantified.